The goal of the research program is to understand the physical- chemical mechanisms of genomic regulation, mediated by the cooperative interactions of regulatory proteins and specific sites on DNA. Biophysical studies of the energetics of interaction of the deoR protein with the E. coli deo operon, and of the bacteriophage lambda cI repressor with its binding sites on the lambda operators, OR and OL are proposed. These will use the Quantitative DNase Footprint Titration method. This method is unique in that it resolves individual-site isotherms, such that both intrinsic and cooperative Gibbs energies can be determined for binding to multiple, specific sites on DNA. These are used to develop statistical-mechanical models of the regulatory interactions. This proposal focuses on the chemical mechanisms responsible for site-specific assembly, and for the coupling between the individual, site specific interactions which regulate the transitions of the entire assembly that activate transcription. DeoR binds to three operator sites, located hundreds of base pairs apart. Full regulation of deo requires additional interactions with the CRP and cytR proteins. Proposed studies will address cooperative coupling, both homologous and heterologous, in this system. Studies conducted at varying inducer concentrations (deoxyribose-5-P, cAMP, cytidine) will define their roles in regulating both the protein-DNA, and cooperative interactions. The cI repressor dimer binds cooperatively to contiguous sites in OR and 0L. The proposed studies will address the role of repressor polymerization in cooperativity. Also, the interactions of each of the repressor subunits with the different basepair sequences of the operator "half-sites" are coupled through the monomer-monomer interface. A combination of protein self-association studies, using analytical gel permeation chromatography, and direct binding studies is proposed, to probe this coupling. The cI repressor uses electrostatic interactions (in part) to distinguish its different operator sites. Studies of the pH and ion linked effects on cI repressor binding, to both operator and non-specific DNA are proposed, to define the role of these forces in the mechanism of site specificity. The energetic properties will be correlated with the known structures of the interacting macromolecules to delineate the roles of individual structural elements in generating the forces.